Breast cancer is a heterogeneous disease with at least 4 major molecularly defined sub-types. Of the >200,000 women diagnosed with breast cancer annually, between 15-25% are diagnosed with triple negative breast cancer (TNBC) which lack estrogen and progesterone receptors and do not exhibit amplification of Her2. These tumors usually have basal-like gene expression signatures and represent the most aggressive and lethal subtype of the disease with few treatment options. During the previous funding period we demonstrated that 1,25D, the active form of vitamin D, markedly suppresses the expression and activity of hyaluronan synthase-2 (HAS2), a gene that is preferentially overexpressed in TNBC. Importantly, women with TNBC whose tumors overexpress HAS2 have significantly reduced survival, suggesting that targeting this gene is likely to have an impact on disease progression. HAS2 encodes an enzyme that produces hyaluronic acid (HA) a secreted polymer that activates the cell surface receptor CD44 which has been functionally associated with the acquisition of stem-cell or tumor initiating cell properties. There is considerable evidence tht TNBCs are dependent on CD44 for survival, but the role of HAS2 and HA in mediating these effects have not been studied. This project will test the hypothesis that TNBCs are dependent on HAS2-generated HA to drive CD44 mediated survival signaling. Our preliminary data strongly suggests that vitamin D suppression of HAS2 activity and HA synthesis represents a feasible approach for interrupting CD44 signaling in TNBC cells. The proposed studies will examine the independent and interactive effects of vitamin D and three other natural products (4-methylumberellifone [4MU], sulfoquinovose [SQ] and glucosamine) on HA metabolism and CD44 signaling in TNBC models in vitro and in vivo. If our pre-clinical studies demonstrate that any or all of these agents impact on disease progression, we propose that measuring biochemical measures of HA metabolism could become a useful screen to identify breast cancer patients who are most likely to respond to these interventions. Completion of this project will thus provide important translational information regarding the use of these agents in women living with TNBC.